A microwell array device capable of measuring single-cell oxygen consumption rates

Molter, T.W.; McQuaide, Sarah C.; Suchorolski, Martin T.; Strovas, Tim; Burgess, Lloyd W.; Meldrum, Deirdre R.; Lidstrom, Mary E.

doi:10.1016/j.snb.2008.10.036

Cited by 77 publications

(97 citation statements)

References 45 publications

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“…The available experimental approaches for oxygen consumption measurements in individual cells can be divided into two groups: one set of techniques is based on microprobes, [18][19][20][21][22] whereas the second involves the isolation of individual cells in microwells. [23][24][25][26] Although microprobe methods provide a robust and sensitive tool for oxygen flux measurements at the singlecell level, they suffer from several limitations, including low throughput (one cell at a time) and the inability to separate potential contributions from adjacent cells in high-density cultures.…”

Section: Introductionmentioning

confidence: 99%

“…27 A different approach for measuring oxygen consumption rates of individual cells has been developed by our group and is based on the measurements of oxygen fluxes in isolated hermetically sealed microwells containing single cells. [23][24][25][26] In contrast to microprobe-based oxygen consumption measurements, methods based on isolation of individual cells in microwells offer at least one order of magnitude higher throughput and true single-cell measurement capability. Enclosing individual cells in hermetically sealed chambers provides a unique possibility to measure isolated oxygen consumption with true single-cell resolution.…”

Section: Introductionmentioning

confidence: 99%

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Method for physiologic phenotype characterization at the single-cell level in non-interacting and interacting cells

et al. 2012

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Abstract. Intercellular heterogeneity is a key factor in a variety of core cellular processes including proliferation, stimulus response, carcinogenesis, and drug resistance. However, cell-to-cell variability studies at the single-cell level have been hampered by the lack of enabling experimental techniques. We present a measurement platform that features the capability to quantify oxygen consumption rates of individual, non-interacting and interacting cells under normoxic and hypoxic conditions. It is based on real-time concentration measurements of metabolites of interest by means of extracellular optical sensors in cell-isolating microwells of subnanoliter volume. We present the results of a series of measurements of oxygen consumption rates (OCRs) of individual non-interacting and interacting human epithelial cells. We measured the effects of cell-to-cell interactions by using the system's capability to isolate two and three cells in a single well. The major advantages of the approach are: 1. ratiometric, intensity-based characterization of the metabolic phenotype at the single-cell level, 2. minimal invasiveness due to the distant positioning of sensors, and 3. ability to study the effects of cell-cell interactions on cellular respiration rates.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Method for physiologic phenotype characterization at the single-cell level in non-interacting and interacting cells

et al. 2012

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“…Extracting this information is difficult. Efforts are being made with single-cell observatories designed to trap cells in microwells and monitor their consumption of oxygen over time using optical techniques (6,7). Field-based approaches involve modeling oxygen pore water profiles in marine sediments and dividing oxygen consumption rate by cell numbers (8).…”

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confidence: 99%

Oxygen Consumption Rates of Bacteria under Nutrient-Limited Conditions

Riedel

Berelson

Nealson

et al. 2013

Appl Environ Microbiol

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bMany environments on Earth experience nutrient limitation and as a result have nongrowing or very slowly growing bacterial populations. To better understand bacterial respiration under environmentally relevant conditions, the effect of nutrient limitation on respiration rates of heterotrophic bacteria was measured. The oxygen consumption and population density of batch cultures of Escherichia coli K-12, Shewanella oneidensis MR-1, and Marinobacter aquaeolei VT8 were tracked for up to 200 days. The oxygen consumption per CFU (Q O2 ) declined by more than 2 orders of magnitude for all three strains as they transitioned from nutrient-abundant log-phase growth to the nutrient-limited early stationary phase. The large reduction in Q O2 from growth to stationary phase suggests that nutrient availability is an important factor in considering environmental respiration rates. Following the death phase, during the long-term stationary phase (LTSP), Q O2 values of the surviving population increased with time and more cells were respiring than formed colonies. Within the respiring population, a subpopulation of highly respiring cells increased in abundance with time. Apparently, as cells enter LTSP, there is a viable but not culturable population whose bulk community and per cell respiration rates are dynamic. This result has a bearing on how minimal energy requirements are met, especially in nutrient-limited environments. The minimal Q O2 rates support the extension of Kleiber's law to the mass of a bacterium (100-fg range). Heterotrophic bacterial respiration constitutes 50% to 90% of ocean community respiration (1) and plays a critical role in the recycling of organic carbon in all natural environments (2). Despite this importance, respiration remains poorly quantified in ocean models of metabolism, gas exchange, and carbon mass balances (3). This uncertainty in heterotrophy makes it difficult to predict when areas of the ocean will be sources or sinks of CO 2 (4). Microbial community ecosystem behavior is ultimately a function of auto-and heterotrophic relationships; hence, understanding the biogeochemical contribution of heterotrophic bacteria at the single-cell level is necessary for making predictions of how ecosystems will respond to global change (5). Extracting this information is difficult. Efforts are being made with single-cell observatories designed to trap cells in microwells and monitor their consumption of oxygen over time using optical techniques (6, 7). Field-based approaches involve modeling oxygen pore water profiles in marine sediments and dividing oxygen consumption rate by cell numbers (8). A more frequently employed method for analyzing cellular respiration rates is to determine bulk oxygen uptake (consumption) rates (OUR), using either batch or continuous cultures with large populations of microbes. The average cellular respiration rate is calculated by normalizing OUR to population size. Most studies of this nature determine respiration rates while cells are growing (9-14). In the rare cases wher...

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“…[5][6][7][8][9][10][11][12][13][14][15][16] Quantification of oxygen concentrations with oxygen-sensitive photoluminescent dyes is measured by either the degree of luminescent intensity quenching [5][6][7][8][9] or the luminescent lifetime. [10][11][12][13][14][15][16] Detecting the luminescent lifetime to quantify oxygen concentrations has been proven to have a higher sensitivity due to the inherent stability of the signal. Thorsen et al proposed a low-cost platform based on phasedomain lifetime detection using a modulated light-emitting diode (LED)-based optical excitation and a detection system for real-time sensing of aerobic and anaerobic bacteria.…”

Section: Introductionmentioning

confidence: 99%

“…Instead, Molter et al proposed a microwell device for measuring oxygen consumption rates where a glass lid attached to the end of a piston is manually pressed onto a single microwell array to diffusionally isolate the single cells in an array of $80 pl microwells using luminescent oxygen sensors. 13,14 The lid blocks any diffusion of oxygen into or out of the microwell containing the cells. However, the manual mechanical actuator required to diffusionally isolate the microwells is massive, complicated, and not conducive for miniaturization into a lab-on-a-chip device.…”

Section: Introductionmentioning

confidence: 99%

Light-addressable measurements of cellular oxygen consumption rates in microwell arrays based on phase-based phosphorescence lifetime detection

Huang

Hsu

et al. 2012

Biomicrofluidics

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A digital light modulation system that utilizes a modified commercial digital micromirror device (DMD) projector, which is equipped with a UV light-emitting diode as a light modulation source, has been developed to spatially direct excited light toward a microwell array device to detect the oxygen consumption rate (OCR) of single cells via phase-based phosphorescence lifetime detection. The microwell array device is composed of a combination of two components: an array of glass microwells containing Pt(II) octaethylporphine (PtOEP) as the oxygensensitive luminescent layer and a microfluidic module with pneumatically actuated glass lids set above the microwells to controllably seal the microwells of interest. By controlling the illumination pattern on the DMD, the modulated excitation light can be spatially projected to only excite the sealed microwell for cellular OCR measurements. The OCR of baby hamster kidney-21 fibroblast cells cultivated on the PtOEP layer within a sealed microwell has been successfully measured at 104 6 2.96 amol s À1 cell À1. Repeatable and consistent measurements indicate that the oxygen measurements did not adversely affect the physiological state of the measured cells. The OCR of the cells exhibited a good linear relationship with the diameter of the microwells, ranging from 400 to 1000 lm and containing approximately 480 to 1200 cells within a microwell. In addition, the OCR variation of single cells in situ infected by Dengue virus with a different multiplicity of infection was also successfully measured in real-time. This proposed platform provides the potential for a wide range of biological applications in cell-based biosensing, toxicology, and drug discovery. V C 2012 American Institute of Physics.

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A microwell array device capable of measuring single-cell oxygen consumption rates

Cited by 77 publications

References 45 publications

Method for physiologic phenotype characterization at the single-cell level in non-interacting and interacting cells

Method for physiologic phenotype characterization at the single-cell level in non-interacting and interacting cells

Oxygen Consumption Rates of Bacteria under Nutrient-Limited Conditions

Light-addressable measurements of cellular oxygen consumption rates in microwell arrays based on phase-based phosphorescence lifetime detection

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